Using measurement or analysis technologies based on probe microscopy, a specimen to be examined is examined experimentally by detecting an influencing of a measurable property of a measurement probe by the specimen to be examined. During the examination of the specimen by means of scanning probe microscopy (SPM), a scanning of the specimen takes place by means of a relative movement between the measurement probe and the specimen. By way of example, a surface for analysis on the specimen is scanned.
One method of scanning probe microscopy is atomic force microscopy (AFM). This serves for measuring surface properties of the specimen, for example the topography, with a high lateral and vertical resolution. The term “lateral resolution” here denotes the resolution in a plane of the surface to be examined. The direction perpendicular to this plane is referred to as the vertical direction. In the vertical direction, the topography of the surface is determined with a vertical resolution. However, volume properties close to the surface can also be determined, for example the elasticity of a specimen. The resolution of the vertical and/or horizontal displacement of the measurement probe relative to the specimen during probe microscopy usually lies in the range of a few nanometers and above, whereas customary methods used for the optical examination of a specimen operate with a resolution of several hundred nanometers.
In order to be able to carry out scanning probe microscopy, the distance between a measurement probe and the specimen to be examined must be able to be set and measured in a very precise manner. As the measurement probe, measurement beams (also known as cantilevers) are used for example in connection with scanning probe microscopes. Particularly in atomic force microscopes, the measurement parameter evaluated is a force occurring as a result of an interaction between the cantilever and the specimen to be examined, it being possible in the simplest case for the force to be described by a Lenard-Jones potential. Several options may be used for detecting the force. In the simplest case, a deflection of the measurement probe is detected, said measurement probe in the case of the cantilever in atomic force microscopy usually being designed as a thin spring beam. However, measurement methods also exist in which the cantilever is made to vibrate. The attenuation of the amplitude of the induced vibration is then the measurement parameter used for the adjustment. One common feature of the known measurement methods is that an interaction between the measurement probe and the specimen to be examined is measured. The term “scanning probe microscopy” in the meaning used here encompasses, in addition to the aforementioned techniques, all techniques in which one or more elements acting as measurement probes scan an object structure.
In one known measurement method, the force acting on the cantilever is detected using a light pointer principle. In this case, a measurement light beam, in particular a laser beam, is directed onto the cantilever, the intention being for focussing to be provided. However, when using the light pointer principle, the problem occurs that the laser spot focussed onto the cantilever should be smaller than the width of the cantilever, in order to allow optimal detection of the bending of the cantilever. Therefore, it must be possible to produce such a small spot using suitable optics. Depending on a bending of the cantilever, the measurement light beam is reflected at the cantilever or at a component connected to the cantilever, at a certain angle relative to the direction of impingement. The reflected light beam is then directed onto a photodiode, which has a receiver surface comprising at least two segments. A difference of the received light beam for the two segments indicates that the measurement light beam is removed from a central position between the two segments. The central position is defined in such a way that the measurement light beam impinges equally on the two segments. A bending of the cantilever leads to the situation that the equal distribution of the reflected measurement light beam across the two segments is changed.
If a torsion of the measurement probe, preferably designed as a cantilever, is also to be detected in addition, use may be made of a photodiode comprising four segments. The use of a different position-sensitive detector may also be provided. As a result, a position determination of the measurement light beam in two dimensions is then possible. Knowing a spring constant of the cantilever, the measurement of the bending of the cantilever can then be used to determine the force between the cantilever and the specimen to be measured.
When scanning the specimen to be measured, usually the distance between the specimen and the cantilever in the vertical direction is set in an exact manner by means of a relative movement of the specimen and cantilever. It is thus possible, for example, to set a constant force ratio. In order to set the distance, use may be made for example of piezoelectric components. At the same time, during the measurement, a scanning-type relative movement of the cantilever laterally to the specimen is carried out.
In principle, either the specimen or the cantilever can be moved by means of displacement or adjustment elements. A splitting of the movement is also possible, for example a lateral movement of the specimen and a vertical movement by the cantilever.
The efficiency of a cantilever-based scanning probe microscope is not least also determined by the cantilever used. On the one hand, the force exerted on the specimen is determined as a function of the deflection via the force constant of the bending beam. For biological specimens, a low force constant is essential so as not to damage the specimen as a result of excessive forces during the measurement. On the other hand, the frequency of the intrinsic resonance and also the quality determine the time in which the cantilever can react to external force changes. The higher the resonant frequency of the cantilever, the quicker the reaction of the bending beam and the quicker scanning can be carried out. Furthermore, the thermally induced noise of the cantilever in a fixed bandwidth is then also reduced. The use of short, narrow and thin bending beams, which are also referred to as “small cantilevers”, is therefore advantageous since in this case a high resonant frequency can be combined with an acceptably low force constant.
Nearfield microscopy (SNOM—“Scanning Nearfield Optical Microscopy”), a sub-type of scanning probe microscopy, is capable of measuring additional optical parameters. In this case, the achievable optical resolution is approximately one to two orders of magnitude better than that of farfield optical measurements, which can be carried out for example using a light microscope. This resolution is achieved by illuminating the specimen for example through an aperture, the diameter of which is less than half the wavelength of the emitted light, or else by collecting through this aperture any light scattered by the specimen. The aperture diameter is in this case the parameter which determines the resolution. As an alternative to a small aperture, a suitable scattering object with a small expanse can also act as the nearfield source, for example the tip of a cantilever of an atomic force microscope. The optical parameters are only measured on the surface in the vicinity of the measurement probe, since the nearfield, due to its nature, has already decayed exponentially to very low intensities after a few tens of nanometers. During the nearfield optical measurement, therefore, the distance between the aperture/scattering object and the specimen must be kept constant in the nm range. A combination of SNOM and force/attenuation-measuring methods such as atomic force microscopy has proven useful for this.
The illumination of the small aperture is usually carried out by a second laser, and therefore the SPM structure should allow the illumination of a nearfield aperture in the cantilever tip by a second laser.
It is often useful to examine the specimen to be examined by means of an optical microscope in addition to the analysis by probe microscopy. This allows an overview of the specimen in a larger field of vision than in the case of scanning probe microscopy. Moreover, such optical microscope examinations are quick and are established as standard methods. In this case, in order to obtain optimized results in the transmitted light or reflected light mode, the specimen must be illuminated by a condenser illumination so as to have sufficient illumination of the specimen for examination by means of a light microscope.
During the optical microscopy, use may be made of various optical contrast methods, such as for example phase contrast or differential interference contrast (DIC), which offer a high resolution and require for optimal evaluation capability a sufficient condenser illumination in the specimen plane, for example with regard to the intensity distribution, polarization distribution, angle of incidence distribution or position of the exit pupil and exit window.
One very simple and generally recognized implementation of a nearfield arrangement is for example to use as the nearfield source a bent optical fibre which is tapered at the end and vapour-coated with a metal layer apart from a small aperture, and simply to introduce it directly over the specimen into the path of the condenser light, which is also referred to as the condenser light path. The specimen thus remains fully accessible to the condenser light apart from a minimal restriction and can be imaged by means of the nearfield microscope without any appreciable restriction of the optical examination method. However, due to the special distance detection (“shear force mode”) used therein, the fibreglass probes cannot be used or can be used only unsatisfactorily for measurements in liquids.
A second possibility for combined examination by means of probe microscopy and optical microscopy is to integrate the scanning probe microscope in an arrangement with an upright optical microscope, which is also referred to as a light microscope. In this case, instead of a normal microscope objective, use is made of special objective which carries the cantilever and can optionally also be displaced in the μm range in up to three dimensions. However, this implementation has the considerable disadvantage that the lack of stability of the upright microscope in the nm range does not allow a high-resolution measurement using the “objective” for atomic force microscopy, due to a tendency of the microscope to vibrate.
One possibility for transmitting the condenser light emitted by the condenser illumination consists in arranging a free central light path, which optionally leads only through planar optical components such as glass plates, beam splitter cubes or the like and thus influences the propagation of the condenser light only to a minimal degree. However, in this case, only the space defined by the working distance of the condenser illumination between the outlet of the condenser illumination and the objective of the optical microscope can be used as installation space for the probe microscope, so that usually only condenser illuminations with a relatively small numerical aperture for the illumination and a large working distance are used.